Ml ICHIGAN STATEU I I IIII I III IIIIIIIIIIIIIIIIIIIIIIIII 3 12 01591 4355 a: :3 LIBRARY ”‘1 MIG i;::m u x .2: :5.‘ Ufllv'fl“ g}. i -- w-‘- -m.~—-v~-m--. "-ku - THFS‘ 3 II This is to certify that the thesis entitled INHERITANCE OF RESISTANCE TO DROP (SCLEROTINIA SCLEROTIORUM) IN LETTUCE presented by Abdul Madjid has been accepted towards fulfillment of the requirements for Ph.D. degree in Horticulture W W Major professor Date April 7, 1981 0-7639 OVERDUE FINES: 25¢ per W per item . RETURN! LI RARY MATERIALS: 1533“,}? ' Place in book return to remove . 1 ‘WI” ., charge from circulation records ‘N‘ I ’IflflN\ L. 1 II‘ . INHERITANCE OP RESISTANCE TO DROP (SCLEROTINIA SCLEROTIORUM) IN LETTUCE BY Abdul Madjid A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of - DOCTOR OF PHILOSOPHY Department of Horticulture 1981 ABSTRACT INHERITANCE OF RESISTANCE TO DROP (SCLEROTINIA SCLEROTIORUM) IN LETTUCE BY Abdul Madjid Using a large number of seedlings, it was possible to screen for resistance to lettuce drop caused by (Sclerotinia sclerotiorum under greenhouse conditions. Reliable results. were obtained by placing 8 cm2 of colonized agar block 1 cm from the base of 18 to 24 day old seedlings. Since the degree of resistance among cultivars varied with the time of observation, resistance was measured by the number of days required to reach 50% mortality and by the number of plants that survived in each cultivar. Culti- vars and accessions that were found significantly higher in level of resistance than Grand Rapids at 9, 12 and 15 days following innoculation were PI 250427, Taiwan, PI 251790, PI 255568, MSU 73-44 and Bibb. A correlation coefficient of r =.77 significant at P = .001 was found between the plants grown in the green- house ground bed and those grown in flats, suggesting that similar results were obtained by both growing methods. A study of inheritance of resistance to lettuce drop was made by using a large number of seedlings from various Abdul Madjid generations involving nine crosses using the following parents:' Grand Rapids, PI 250427, PI 278110, and 4 TaiWan strains. Variation in the time required to reach 50% mor- tality was observed to be primarily due to environmental effects, although low genetic variation for resistance was noted based on (1) variation in survival percentage as ob— served in the parents, and (2) genetic gain that was evident in the F3 generation when a large number of lines were sam- pled: and no gain was observed when a low number of lines were sampled in the F4 generation. These results suggest that resistance to lettuce drop in the cultivars studied was polygenically controlled with low level of resistance. The possibility of obtaining a greenhouse cultivar resistant to lettuce drop is discussed. DEDICATION To my wife, Lilis Isminatun, for her understanding and encouragement, and To my children Winarto, Yulia, Dedi, Suzy and Dewi, and To my niece, Yati, for their understanding ii ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr. Shigami Honma for his guidance throughout the study. I also wish to acknowledge the members of my guidance committee, Dr. Larry Baker, Dr. Jon Fobes, Dr. Robert Herner, Dr. Melvin Lacy, Dr. Hugh Price and Dr. Jonathan wright. Special thanks to George Storm, Gene Mero, Kenneth McCammon and Vince Fritz for their assistance in conducting the experiments, and to Mrs. LuAnn Kent for any help she has given. I wish to express my grateful thanks to Ir. Sadikin Sumintawikarta, Head of the Agency for Agricultural Research and DeveIOpment for awarding me the fellowship, and to Mr. Joseph R. Bookmyer and Dr. Edwin B. Oyer for their best effort in managing the fellowship. I also express my special appreciation to my wife, Lilis, my daughters Suzy and Dewi for their help in many ways. iii TABLE List of Tables. . . . . . . . . List of Figures . . . . . . . . INTRODUCTION . . . . . . . . . REVIEW OF LITERATURE. . . . . . The Pathogen . . . . . . Screening Techniques . . 1. Age of Plants. . 2. Type of Inocula. 3. Optimum Time for Disease Resistance . . . Penetrance . . . . . . . PART I. SCREENING TECHNIQUES . MATERIALS AND METHODS. . Plant Materials. . . Age of Plants . . . Types of Inocula . . Time of Evaluation . Relationship Between Culture. . . . . . . OF CONTENTS Cultivar Ev Bed and Statistical Interpretation . RESULTS AND DISCUSSION . Effect of Age of Seedlings on Mycelial suspensions and luation 1. Disease Incidence. .. 2. Types of Inocula a. Sclerotia . b. blocks . . . 3. The Optimum Time for Cultivar Evaluation . . . 4.. Bed vs. CONCLUSIONS. . . . . . . PART II: MATERIALS AND METHODS. iv RESISTANCE TO LETTUCE DROP Experiment in the Greenhouse Using Flats. vi viii 29 31 35 35 Parental Materials. . Pollination . . . . . QUIhWNl-J O O Populations . . . . . 7. Genetic Interpretation. . . RESULTS AND DISCUSSION. . . . Parental Materials. . . . Pooling of the Data . . . Inheritance of Resistance Grand Rapids Crosses. PI 278110 Crosses . . F and P Generations Igterprefation of the CONCLUSIONS . . . . . . . . . APPENDIX . . . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . Screening for Disease Resistance. Screening of the F Population Screening of the F Population. Weighting of the Backcross .35 .36 .39 .41 .42 .43 .44 .45 .45 .45 .47 .47 .51 .52 .55 .58 .60 .64 10. ll. 12. 13. LIST OF TABLES Lettuce cultivars and accessions used in the study of screening techniques. . . . . . . . . . . . . . .15 Statistics utilized to calculate homogeneity test of regression coefficient. . . . . . . . . . . . . .22 The effects of seedling age and inoculation tech- nique on percent seedling mortality at 10 days after inoculation. . . . . . . . . . . . . . . . . .24 The effect of inoculation technique on percent mortality. 0 O O O O O O O O O O O O O O O O O I O .26 Variance analysis for the effects of inoculation teChniques O O O O O O O O O O O O O O O O O O O O O 26 Slopes (b), intercept (a) and correlation coef- ficient (r) for cumulative number of mortality of lettuce at 5 observations after inoculation . . .27 Number of days when 50% of plants died and survival percentage at 15 days after inoculation for 16 cultivars. . . . . . . . . . . . . . . . . . . . . .28 Correlation between percentages of surviving plants grown in the greenhouse ground bed and those grown in flats in the same greenhouse. . . . . . . . . . .30 Number of the F lines screened for resistance to lettuce drop 0 C O O O O O O O O O O O O O O O O O O 40 The soil and air temperatures in the greenhouse during each of the six plantings . . . . . . . . . .42 Number of days after inoculation to reach 50% mortality and percent survivors on the 6th day following inoculation of the parent lines. . . . . .46 Time of observation to reach 50% mortality for Grand Rapids crosses . . . . . . . . . . . . . . . .49 Survival percentage at third and last observations for Grand Rapids crosses . . . . . . . . . . . . . .50 vi TABLE Page 14. Time of observation 50% mortality for PI 278110 crosses. . . . . . . . . . . . . . . . 52 15. Survival percentage at third and last ob- servations for PI 278110... . . . . . . . . . 53 16. Mean survival percentages in the F and F lines compared to the parental meags and F , for Grand Rapids crosses. Parental means 2 100% . . . . . . . . . . . . . . . . 54 4 17. Mean survival percentages in the F3 and F4 lines compared to the parent means and F2 for PI 278110 crosses. Parental Means = 100% 56 18. Number of lines the F and F generations having significantly greater survival per— centage than that of the parent means . . . . 56 APPENDIX TABLES A-1.Survival percentage for 16 cultivars ob- served at several number of days following inoculation . . . . . . . . . . . . . . . . . 60 A-2.Survival percentages of the parent materials observed at different number of days following inoculation . . . . . . . . . . . . . . . . . 61 A-3.Frequency distribution of the number of dead plants at different times of observation from S crosses involving Grand Rapids. . . . . . . 62 A-4.Frequency distribution of the number of dead plants at different times of observation from 4 crosses involving PI 278110 . . . . . . . . 63 LIST OF FIGURES FIGURE PAGE 1. Resistance and collapsed susceptible plants 15 days after inoculation. . . . . . . . . . . . l9 2. Parental materials . . . . . . . . . . . . . . . 38 viii INTRODUCTION Lettuce drop is a disease of greenhouse and field lettuce caused by Sclerotinia sclerotiorum (Libert) de Bary. The fungus attacks various other species of crops and weeds in the field, and also causes decay in trans- ported and stored lettuce. The fungus attacks the lettuce at the base of the plant, girdling the stem and causing the plant to collapse or drop. The fungus survives in a dormant state as sclerotia, which are able to withstand extreme variation in environmen- tal conditions. Sclerotia under cool and moist conditions may germinate, either forming mycelia or apothecia which release ascospores. Ascospores may be the source of primary infection in the spring. The wide host range of Sclerotinia, its ability to grow saprophytically, and the ability of the sclerotia to survive long periods of time under extreme en- vironmental conditions, help make lettuce drop an important disease of lettuce. Several control measures have been attempted (flooding the field, crop rotation, chemical control, etc.) with varying degrees of success. Field resistance was reported on several accessions of lettuce (Newton and Sequeira, 1972b). Under greenhouse conditions, however, these accessions were found 2 to be susceptible. Knowledge on the inheritance of lettuce drop resistance would be valuable for breeding for disease resistance in greenhouse lettuce. The purpose of this study was to determine a simple and reliable Screening technique for lettuce drop and to study the inheritance of resistance to lettuce drop. REVIEW OF LITERATURE The Pathogen The genus Sclerotinia was originally established by Fuckel in 1870 covering 5 species, 4 of which are known to be important as a plant pathogen (Korf and Dumont, 1972). A new generic name Whetzelinia sclerotiorum (Libert) Korf and Dumont (Korf and Dumont, 1972) was recently proposed for Sclerotinia sclerotiorum (Libert) de Bary. A proposal to preserve the previous generic name, however, has been ac- ,cepted by the International Association of Plant Taxonomists (Kohn, 1979), and therefore the generic name Sclerotinia is retained. According to Adams 3: 31., (1974) the fungus attacks 190 crop plants and weed species in 130 genera and 45 fam- ilies of plants. Schwartz (Schwartz gt 21.,1978) mentioned a host range covering 374 species of 237 genera in 62 plant families. In vegetable crops, Chupp and Sherf (1960) listed 53 crops which are attacked by the disease. They also listed the different common names of the pathogen when it infests various hosts. The common name for the disease on lettuce is lettuce drop: it is less commonly known as collar rot. The rot begins at the stem near the soil surface and spreads downward towards the roots. The peticles decay, starting from the point of attachment to the stem upward, causing the leaves to drop. The fungus then spreads over all collapsed leaves. Cottony mycelia covers the decayed plant parts. The sclerotial initials were white, the sclerotia then become brown and finally black. The fungus is known to be adaptable to a wide range of environmental conditions. The most favorable temperature for infection in lettuce is about 10-25 C (Abawiand Grogan, 1979), but the optimal growth in litre ranges from 20 C (Tanrikut and Vaughan, 1951) to 25 C (Bedi, 1962). The fungus grows best under abundant moisture from rain, fog, or sprinkler irrigation (Chupp and Sherf, 1960). Prolonged soil flooding, however, kills the sclerotia (Moore, 1949). Primary infection by S. sclerotiorum results from infection by ascospores produced by apothecia (Natty, 1971; Newton and Sequeira, 1972a) and mycelia from sclerotia. Under natural conditions at temperatures of 9-17 C for 14 days, apothecia often arise from sclerotia (Letham 25 31., 1976). Under natural conditions, plant canopy character— istics were found to have direct effects on the types of inoculum formed. In cauliflower or lettuce, the canopy was dense which prevented the drying of the soil surface. This condition resulted in the inhibition of germination of sclerotia to produce mycelium, but maintained suitable con- ditions for apothecia formation. On the other hand, tomatoes trained on trellies left the soil exposed and dry, which stimulated the sclerotia to produce mycelium. The apothecia remain functional (unshriveled) for about 7 days after germination (Schwartz and Steadman, 1978). A sclerotium under natural conditions produces an average of 2 apothecia. Ascospore spreading is by wind or in the case of rape seed (Stelfox £3 31., 1978) was reported to be spread by bees. When ascospores landed on susceptible plant parts, infection occurred within two days and symptoms appeared in four days (Chupp and Sherf, 1960). Secondary infection of S. sclerotiorum may occur from mycelia grown from sclerotia and from mycelium fragmentation. Adams and Tate (1976) showed that eight weeks after placing the sclerotia on the soil, more than 22% of sclerotia ger- minated. Only two days after germination, the mycelium in- fected the hosts. In the case of S. sclerotiorum minor, the authors found the mycelium directly attacked the let- tuce plants without first colonizing organic matter. The formation of sclerotia by the fungus was apparently affected by temperature as demonstrated by Eedi (1962). Potato Dextrose Agar (PDA) cultures of the fungus produce sclerotia slower and there are fewer sclerotia formed at low temperatures. At a temperature of 25 C sclerotia are formed in four days. Studies by Smith (1972) showed that the sclerotia could be induced to produce mycelia by alter- nate drying and rewetting of the sclerotia. The sclerotia have been known as a potential source of inoculum in soil. The survivability depends on: 6 (a) Weather conditions in summer; dry summers being favorable for survival. (b) Cropping practices; cropping with susceptible crops increases the number of the potential inocula. (c) Depth of sclerotia in the soil. Adams (1975) showed that sclerotia of S. sclerotiorum buried in the field at l, 6 and 12 inch depths sur- vived well over a 15 month period, but it sur- vived poorly at a depth of 24 inches. Plant debris in the field under favorable conditions _ (cool and moist) can be covered by lettuce drop mycelia with- in 5 days. Tanrikut and Vaughan (1951), supported by Purdy and Grogan (1954), postulate that since S. sclerotiorum is able to grow in limited substrates, it can live for a long period as a saprophyte. This extreme adaptability and habit of producing tough, resistant sclerotia indicate that S. sclerotiorum is a disease that is difficult to control by growing nonsusceptible crops in a rotation. Screening Techniques 1. Age of Plants The effect of age on the susceptibility of lettuce plants to drop has not been carefully studied. Different authors tested various ages of seedlings with several inocula. Adams and Tate (1976) infected 2 week old transplanted seed- lings of lettuce of sclerotia of S. sclerotiorum minor. Six percent of the sclerotia were found to germinate and cause infection. Using mycelia in agar blocks, Newton and Sequeira (1972b) found that inoculating 28 day old plants in the‘ greenhouse killed all of the seedlings. The effect of stage of growth on infection by asco- spores in bean is remarkable. Ascospore inoculation has been successful when applied to blossoms, but not to leaves (Abawi, 23 31., 1975). 2. Type of Inocula Inocula used to study pathogenicity of S. sclerotiorum have been either sclerotia, mycelia or ascospores. The use of sclerotia on lettuce by Adams and Tate (1976) has been referred to earlier. These authors found that germination of sclerotia were observed only on the small sclerotial isolates, but not on the large isolates. Adams (1975) re- ported that using sclerotia produced an oat seed media gave 90% infestation on Romaine and head lettuce (Mesa 659), but did not infest escarole (Cichorium endivia). Abawi and Grogan (1975) demonstrated that in bean, mycelium from sclerotia infected bean readily when an available source of energy such as dead or senescent tissues were found in direct contact between sclerotiaemuithe bean stem. The need of the presence of non—living organic matter to initiate infection by mycelia from sclerotia was confirmed in lettuce study (Purdy and Grogan, 1952). No infection was observed when dead lower leaves were removed, but infection occurred when sclerotia were placed at 6 mm from the stem and covered with dead leaves which touched the stem. These findings suggest that sclerotia do not provide sufficient nutrient for mycelial development in plant. Using a 1.0 cm diameter agar blocks of PDA culture or infected oat kernel placed near the base of the plants, Newton and Sequeira (1972b) found that 2 to 3 seedlings of each field resistant line of lettuce died within 2 to 4 days when inoculated with colonized oat seeds or agar blocks in the greenhouse. However, field inoculation with . residue of the infected plants resulted no infection in the field for the lines PI 184787, PI 187239, PI 250427, PI 251790 and PI 255568. When mycelial inoculation was done in the field, all lines died except PI 184787, PI 165063, PI 250427, pr 250429 and PI 255568. The difference in the results was probably due to succulence, low fiber content and undetermined factors found in plants grown in the green- house. Kreitlow (1951) working with Ladino clover did not find satisfactory results by scattering pieces of agar cul- ture on the soil among the plants grown in flats. The in— fection was not uniform and the agar was quickly overgrown by contaminants. The use of grain inoculum in studying pathogenicity of S. sclerotiorum on several species of plants was reported by Price and Colhoun (1975b). Using S. trifoliorum, Kreitlow (1951) obtained good results in the field and greenhouse tests by applying moist infected grain to clover. Suscep- tible plants in the greenhouse were killed in 5 to 15 days following the inoculation. The disadvantage of applying moist infected grain was the wet grain tended to lump and was difficult to apply evenly to individual seelings. Mycelia of S. trifoliorum inoculated by spraying was studied by Frandsen (1946) on pasture legumes. The suspen- sion was preparedlnrmixing the culture with clover decoction. One dosage consisted of 5 plates of mycelia 5-6 cm diameter suspended in 1 liter of clover decoction for 8 flats of 44 x 40x 15 cm. The number of dead plants found varied with the different concentration of inocula, gig. 83%, 79.5% and 68.5% with the concentration at 1%, 1 and 8 dosage, respect- ively. S. sclerotiorum does not form conidia, it produces ascospores liberated from apothecia. According to Kreitlow (1951). it is difficult to secure adequate numbers of asco- spores for large scale plant inoculations. The simplest technique in producing apothecia was reported by Bedi (1956). It consisted of floating the sclerotia in water for approx- imately 6 weeks at 15-20 C. But this technique, and several other techniques developed by different authors, when tested by Price and Colhoun (1975a) did not result in mature apo- thecia formation, although stipes were formed. In addition, since the viability of ascospores is limited to 4 days 10 (Newton and Sequeira, 1972a), a continuous supply of mature apothecia would be needed during the inoculation studies. 3. Optimum Time for Cultivar Evaluation Infection of lettuce plants inoculated with sclerotia of S. sclerotiorum begins 2 days after inoculation (Adams and Tate, 1976). The percentage of plants infected increased and reached its maximum 14 days following inoculation, and remained the same when observation was ceased 22 days fol- lowing inoculation. In several generations of tomato crosses which were assessed for resistance to tobacco mosaic virus, the degree of resistance changed with the age of the plants (Phillip 35 31., 1965). Similar results were found in evaluating host resistance to Stewart's disease (Erwinia stewartii) in corn (Blanco £3 31., 1979). From data collected at 3 different times of observation, the authors were able to differentiate lines contributing genes for suppressed disease development. Disease Resistance Literature on the resistance to lettuce drop is limited. A lettuce cultivar grown in Europe was mentioned by Chupp and Sherf (1960) as almost completely immune to drop. Fifteen cultivars with a high level of field resistance were observed among 125 cultivars tested during 2 summer seasons by Elia and Piglionica (Newton and Sequeira, 1972b). The ll resistance was associated with red pigmentation and leaf type of the cultivars. In field evaluation of 178 lettuce accessions, Newton and Sequeira (1972b) found 21 lines with varying degrees of field resistance. Five accessions (PI 184787, PI 165063, PI 250427, PI 250429 and PI 255568) were found to be most resistant. The nature of resistance was associated with the structure of the plant. The erect type of plant with its open foliage prevented the build up of high relative humidity favorable to disease infestation, while in butter- head and leaf types the resistance was postulated as caused. by less wounding by wind blown particles. Wounding has been considered to predispose lettuce to ascospore infection. The resistance,however, was less when the plants were tested under greenhouse conditions, probably due to more succulence, less fiber content, etc. Based on the breeding tests, the authors believed that the field resistance was genetically inherited; however, no genetic ratio of resistance: sus- ceptibility was reported. Resistance to S. sclerotiorum associated with plant structure was also found on bean (Anderson 33 31. 1960; Coyne 33 31., 1977; and Schwartz 33 31., 1978). A low level of disease incidence was found in cultivars with an open up- right plant habit. Plants with indeterminate habit were likely to be susceptible owing to the heavy canopy. Among the determinate plants, cultivars with a dense leaf canopy 12 were susceptible. Coyne £5 31. (1977) found a high correl- ation between high yielding ability of GN Nebraska and susceptibility to white mold. This high yielding cultivar had a heavy canopy, causing lack of air circulation and light penetration. Dow and Lumsden (1975) found differences in the nature of infection between resistant Scarlet Runner bean lines (£,coccineus) and susceptible P. vulgaris. The resistant tissues acted as a physical barrier for the rapid penetra- tion of the pathogen. Such a barrier was not found in the susceptible tissues of P. vulgaris. Inheritance studies of the resistance showed that the resistance was governed by a single dominant gene (Abawi st 31., 1978), assigned the gene symbol W3. Penetration Interpreting genetic data may be complicated by in- complete penetrance of a character. As defined by Allard (1960), penetrance is the ability of a gene to be repressed in individuals carrying it. The phenomenon was demonstrated in deficient chlorophyll in the tip and leaf margins of unifoliate leaved of lima bean 22° Ventura. Deficiency of chlorophyll is a dominant character, but even in the homo- zygous condition it is rarely found in more than at 10% frequency. The penetrance is approximately 10%. It was reported that under certain environmental conditions the penetrance is complete, while in another condition is 0. 13 In mosquito (Aedes aegypti L.), inheritance studies with the gene Gold affecting mesonotal scale color were inconclusive until Klassen (1964) showed the existence of varying degrees of penetrance for the gene in male and fe- male insects. Incomplete penetrance was suggested in the resistance to Fusarium wilt race I in tomate (Retig gtjfl., 1967). In a backcross with the susceptible parent, 150 healthy: 170 diseased plants were found, suggested 90.6% penetrance in the F1. In the study of resistance to internal browning in tomato, Phillip (1964) found incomplete penetrance in the cultivar Fireball. Resistance was suggested as caused by the interaction of 2 genes, and the level of penetrance was dependent upon the genotype of the plant. PART I: SCREENING TECHNIQUES MATERIALS AND METHODS Plant Materials Ten accessions were utilized in evaluating variable response due to seedling age while 16 accessions were used in evaluating the various inoculation techniques. Under field conditions, PI 165063, PI 184787, PI 187239, PI 250427, PI 250429 and PI 255568 were described as resistant (Table 1), while PI 206965 and PI 251790 were described as tolerant to lettuce drop by Newton and Sequeira (1972b). All Plant Introduction accessions were obtained from the Western Regional Plant Introduction Station, Pullman, Washington. The six cultivars tested represent the four types of lettuce, Lactuca sativa, (leaf, Romaine, butterhead and head types). The seeds of these cultivars were obtained from Joseph Harris Company, Inc., Rochester, New York. To obtain uniform germination, the seeds were sown in the evening in 8 cm deep furrows of moist vermiculite in clay pots. Next morning, the seeds were thinly covered with moist vermiculite. The seedlings were transplanted at the cotyledon leaf stage 7 to 10 days after sowing into steri- lized soil in flats. 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Plants were fertilized 7 and 21 days after transplanting with a solution of 2 g of 20-20-20 (N—P-K) soluble fertilizer per liter of water. Age of Plants In preliminary studies, 140 seedlings from each of 4 cultivars were inoculated as 30 day old plants, died in 7 days following inoculation. When the same cultivars were inoculated at 3 weeks of age a variation in the age at which the plants succumbed to disease was observed. To learn the optimum age for inoculation, plants were inoculated at 18 and 24 days after transplanting. Types of Inocula The culture of S. sclerotiorum used in this study was isolated from lettuce and obtained from Mr. David Willis of the Department of Botany and Plant Pathology, Michigan State University. The inoculum was cultured on potato dextrose agar (PDA) medium and incubated at 20-25 C for 5 days prior to its use. The types of inocula used were: (1) agar block colon- ized with mycelia, hereafter referred to as agar block, (2) mycelial suspension applied as a spray (3) sclerotia. The agar block inoculation technique was adapted from that described by Newton and Sequeira (1972b), by placing 2 either 1 cm or x cm2 blocks of agar, 1 cm from the base of 17 the plant. The mycelial suspensions were prepared by blending 200 ml of water with the equivalent 1 cm2 orlk cm2 of agar block per plant with a blender. One and a half petri dishes of PDA culture were used for 70 plants which approximated 1 cm2 of agar block per plant. Mycelial suspensions using 2 of agar block per plant will cultures equivalent to 1 cm be referred to as full dosage concentration, while that equal to using 8 cm2 of agar block per plant will be referred to as a half dosage concentration. Sclerotia used were obtained from 10 day old agar cultures. The size of sclerotia averaged 3 mm in diameter. The sclerotia were mixed into the soil, 20 sclerotia per flat. The experiments were conducted in the greenhouse in a completely randomized design with 2-10 replicates, and 4-7 seedlings per plot. The total number of seedlings included in the inoculation studies were 1960 plants. Time of Evaluation To study the optimum time for cultivar evaluation, plant stand was recorded every day starting from the sixth day following inoculation and was terminated on the 18th day. A plant was considered as dead when it collapsed or the stem was girdled (Figure l). A total of 512 seedlings from 16 lines were observed in this test. 18 Figure 1: Resistance and collapsed susceptible plants, 15 days after inoculation. 20 Relationship Between Bed and Flat Culture To study the relationship between data obtained from experiments conducted in the greenhouse ground bed and those made in the flats, F2 populations from 5 families were used. These families were derived from crosses between Grand Rapids x PI 250427 and Grand Rapids x 4 Taiwan lines. The F2 seed- lings grown in the greenhouse bed were part of an experiment conducted to study inheritance of resistance to lettuce drop. Materials grown in the flats were grown in the same houses as the greenhouse bed. Seeds for both experiments were germinated in moist vermiculite in the greenhouse. Ten-day-old seedlings were transplanted to 5 x 5 cm peat pots containing artificial medium. The pots were placed in flats, 70 pots per flat. Fertilizer was given 3 and 17 days after transplanting by applying a solution of 2 g of 20-20-20 (N-P-K) soluble fer- tilizer per liter of water. The plot in the flats consisted of 28 seedlings per line, while those in the greenhouse ground bed contained 50 seedlings. Each plot in both planting methods was replicated 3 times in a completely randomized block design. A total of 420 and 750 seedlings respectively were planted from the experiments in the flat and in the bed. Data on the percentage of dead plants were taken 10 days after inoculation. 21 Statistical Interpretation An analysis of variance was carried out for data on age of plants, techniques of inoculation and the optimum time for cultivar evaluation. Regression analysis was con- ducted for cumulative mortality recorded at five observa— tion dates following the inoculation of 16 cultivars. Homo- geneity test for regression coefficient was made following the procedure described by Steel and Torrie (1960) (Table 2). The number of days following inoculation when 50% of plants succumbed was estimated using the following linear regression formula: I y = a + bx, hence x = (y - a)/b where x a the number of days when 50% of the plants died, y a 50% of the total number of the plants that died at the final count, a x intercept of the linear regression, b a slope of the linear regression. Differences in the percent survivors of cultivars tested were analyzed using Tukey's test for significant difference. 22 TABLE 2: STATISTICS UTILIZED To CALCULATE HOMOGENEITY TEST or REGRESSION COEFFICIENT (ADAPTED FROM STEEL AND TORRIE, 1960). Culti- vars n b a SSX SSY SSXY Reduced SS 2 (=b x (=SSY - (SSXY) SSX) /SSX) PI 165063 . . . . . . . Mesa 659 . . . . . . Total(T) TN TSSX TSSY TSSXY T Reduced SS (=A) B=TSSY - (TSSXY)2/TSSX Test for homogeneity F a ((B-A)/(cv-l))/((A/(Tn~2cv)), with df =(cv-l) and (Tn-2cv) RESULTS AND DISCUSSION 1. Effect of Ageof Seedlings on Disease Incidence In an earlier study utilizing 140 seedlings from each of 4 cultivars, 30 day old plants grown in flats succumbed a week after inoculation. Further observations with the same materials showed that when 3-week—old plants were in- oculated, it took a longer period before the plants died. The difference in the length of time for the plants to die was probably due to the dense canopy found in the 30-day-old plants which created a condition more favorable for the rapid development of the fungus. I The optimum seedling age for inoculation was found to be at 18 to 24 days (Table 2). The plants inoculated at 18 days of age showed 69% mortality, while those ino- culated at the age of 24 days had 66% mortality. 2. Types of Inocula a. Sclerotia When sclerotia was used as inocula, none of the plants died (Table 3), although more than 60% of plants inoculated with mycelium died. The results were observed 10 days after inoculation, which probably was too early, Rudorf (1937) found that under favorable conditions, infestation through 23 24 TABLE 3: THE EFFECTS OF SEEDLING AGE AND INOCULATION TECHNI- QUE ON PERCENT SEEDLING MORTALITY AT 10 DAYS AFTER INOCULATION SeedlingfiAge (Days) Inoculation Technique 18 24 Percent Seedling Mortality Control 0 0 Scle otia 0 0 1 cm agar block 1 65 65 full dosage spray 69 73 half dosage spray 75 61 no significant difference between ages of plants, and be- tween agar block and spraying technique. 1equal to 1 cm2 agar block culture per plant sclerotial inoculation was obtained in three weeks. Kreitlow (1951) did not obtain infection with this method although the environmental conditions were favorable for the growth of the mycelium. According to Adams and Tate (1976), dor- mancy sclerotia of each isolate varied. Therefore, it was assumed that the differences in Rudorf and Kreitlow results may be due to differences in length of dormancy period among the isolates used in the studies. Since no attempt was made to use sclerotia from other isolates, the effect of dormancy period could not be confirmed. Results of this study suggested that the use of sclerotia was not satisfactory for evaluating disease re- sistance. 25 b. Mycelial suspensions and agar blocks No significant differences were found between the use of agar block and the mycelial suspension spray, and between the size of agar block. Applying full dosage spray was not statistically different with that of half dosage (Table 3). However, in the second experiment (Table 4), these dosages spray were found significantly different at the 5% level (Table 5). The inconsistency of the results of spraying may have been due to the lowering of the inoculum potential. Although placing the blocks individually next to the plants required more time, the agar block technique was considered as more reliable for screening lettuce cultivars than the spray technique, because the technique insured the same amount of inoculum given. 3. The Optimum Time for Cultivar Evaluation Tests for homogeneity of the regression coefficients for cumulative mortality at 5 different dates following ino- culation for the 16 cultivars did not show significant dif- ferences (5’ 10%). The slopes of regression lines for all the cultivars were similar (Table 6). Cultivar differences were found in the intercept (a) values, suggesting variation in resistance can be measured by differences when the plants from each cultivar died. The number of days when 50% of the plants died for the 16 cultivars ranged from 8.4 to 12.7 days (Table 7). In most of the cultivars this value was between 9 and 12 days and in PI 250427 and Taiwan it exceeded 12 days. 26 TABLE 4: THE EFFECT OF THE INOCULATION TECHNIQUE ON PERCENT OF MORTALITY Inoculation Technique Percent Mortality 1 cm2 agar block per plant 50 0.5 cm2 agar block per plant ' 49 full dosage spray1 53 half dosage spray 432 1equal to 1 cm2 agar block culture per plant 2significantly different at the 5% level from the full dosage spray (LSD .05 = 9%). TABLE 5: VARIANCE ANALYSIS FOR THE EFFECTS OF INOCULATION TECHNIQUES Source of Variance df ms F Total 19 Time 1 47.74 1.96 Blocks 3 114.50 4.69* Treatments 3 103.80 4.25* Error 12 24.41 27 TABLE 6: SLOPES (b), INTERCEPTS (a) AND CORRELATION CO- EFFICIENTS (r) FOR CUMULATIVE NUMBER OF MORTALITY OF LETTUCE PLANTS AT 5 OBSERVATIONS AFTER INOCULA- TION Correlation Cultivars Slopes(b) Intercepts (a) Coefficient(r) PI Accessions: PI 165063 1.63 -5 .97 PI 184787 1.93 -1.8 .92 PI 187239 1.73 - .6 .95 PI 206965 1.73 -l.4 .98 PI 250427 1.97 11.8 .99 PI 250427 1.93 -4.8 .96 PI 251790 1.4 —5 .99 PI 255568 1.7 -7.4 .97 MSU Breeding Lines: Taiwan 7 1.7 11.6 .99 MSU 73-44 1.83 -9 .98 Cultivars: Grand Rapids 2.37 11 .95 Ithaca 1.9 -7 .97 Valmaine Cos 1.8 -3.2 .94 Bibb 1.67 -7.8 .98 Parris Island 1.97 -7.4 .98 Mesa 659 2 -4.4 .94 28 TABLE 7: NUMBER OF DAYS WHEN 50% OF PLANTS DIED AND SURVIVAL PERCENTAGE AT 15 DAYS AFTER INOCULATION FOR 16 CULTIVARS Cultivars Number of Days Survivors*) PI accessions: PI PI PI PI PI PI PI PI MSU Breeding Lines: 165063 184787 187239 206965 250427 250429 251790 255568 Taiwan MSU 73-44 Cultivars: Grand Rapids Ithaca Valmaine Cos Bibb Parris Island Mesa 659 for 50% mortality (%) 10.4 44c 8.7 16a 8.4 l9ab 9.2 25ab 12.1 50e 9.7 25ab 10.7 50e 11.1 47de 12.7 60e 11.5 44c 10.5 19ab 10.3 32de 9.0 22ab 11.3 50e 10.4 28abc 9.5 21ab *) Percentage within column followed by the same letter not significantly different at the 5% level (Tukey's test). 29 If the degree of cultivar resistance was measured from the data obtained at the 12th day, one cannot measure the degree of resistance of the cultivars that survived beyond the 12th day. This is evident since a small number of plants died at the 15th and 18th day (Appendix Table A-l). Since re- sistance is measured from inoculation to death, evaluating resistance should be based on the greatest number of days when 50% of plants succumbed between cultivars. The great- est number of days for 50% mortality was 12.7 (Table 6): therefore evaluation of susceptibility was based on the cumulative number of survivors up to the 15th day following inoculation. Cultivars showing significant differences (P a 0.05) compared to Grand Rapids at 15 days.following inoculation were PI 250427, PI 251790, PI 255568, Taiwan, MSU 73-44 and Bibb (Table 7). Cultivars showing signifi- cant differences from Grand Rapids from the second obser- vation to the fourth observation were PI 250427 and Taiwan (Appendix Table A-l). Experiment in the Greenhouse Ground Bed vs. Using Flats Results of the study on the relationship between greenhouse ground bed XE- flats showed a correlation coefficient of r = .77 (Table 8), suggests that the re- sults from growing in the flats were similar to those in the ground beds. 30 TABLE 8: CORRELATION BETWEEN PERCENTAGES OF SURVIVING PLANTS GROWN IN THE GREENHOUSE GROUND BED AND THOSE GROWN IN FLATS IN THE SAME GREENHOUSE Greenhouse Bed Flats Replicates Replicates F2 Families 1 2 3 l 2 3 Grand Rapids x PI 250427 52 38 50 57 61 57 Grand Rapids x Taiwan-1 53 70 46 89 96 54 Grand Rapids x Taiwan-2 64 60 48 100 64 75 Grand Rapids x Taiwan-3 68 80 66 96 100 82 Grand Rapids x Taiwan-4 56 64 60 86 89 89 r= .77***, P = .001. CONCLUSIONS Ageof Seedlings In a study carried out by Newton and Sequeira (1972b) with PI lines, resistant to Sclerotinia in the field, 2 to 3 plants of each line grown in 15 cm clay pots died within 2 to 4 days when inoculated in the greenhouse at the age of 28 days. Preliminary studies were made in the greenhouse in winter 1978 using 140 30 day-old seedlings of each of the 4 cultivars. Seedlings were grown at 5 x S cm in flats. A11 plants died one week following inoculation using 1 cm2 agar block. When the test was repeated using 3 week old seedlings, there was a variation in time when each of the plants died. The difference between the results obtained between these two plant ages was probably due to the dense canopy noted in the 30 day old seedlings, which provided a more favorable environment at the base of the plants for mycelial development. Such a condition could also have happened with the materials studied by Newton and Sequeira. These authors suggested that under greenhouse conditions, the PI lines lost their tolerance to lettuce drop. No test at different ages was made by these authors. In preliminary studies using a large number of seed- lings inoculated at the let and 30th day after transplanting 31 32 suggest that screening for susceptibility to lettuce drop in the greenhouse is possible by inoculating plants less than 30 days old. When plants were inoculated made using 350 seedlings of 18 and 24 day old plants, no significant difference at the 5% level was noted in mortality for the two age groups. Although statistically not significant, plants incoulated at the 18th day after transplanting showed more dead plants. Mellinger (1968) reported similar observations working with S. sclerotiorum on potato. It appears that the best age for screening for lettuCe drop in the greenhouse is 18 to 24 day old plants. Type of Incoula Results using sclerotia as an inoculum were not satis- factory since it required a longer period (3 to 6 weeks) to obtain infection (Rudorf, 1937), and infection was dependent upon the presence of non-living organic matter which was necessary for the mycelia from sclerotia to initiate in- fection (Purdy and Grogan, 1952). Although 268 seedings were used in this study, no infection was observed 10 days following inoculation on plants grown in sandy soil low in organic matter. The use of mycelium on agar plugs was reported ef- fective in inoculating lettuce grown in the greenhouse (Newton and Sequeira, 1972b). One centimeter diameter agar 33 plug was placed at the base of each plant. In this study, 1/2 cm2 or 1 cm3 of agar block was placed 1 cm from the base of each plant. Both of the blocks were effective. It is suggested 1/2 cm2 of agar block can be used for screening lettuce for disease resistance. Mycelium as a suspension spray toa concentration equal to 1/2cm2 of agar block per plant gave inconsistent results. This is probably due to the lowering of the inoculum potential. Although the placing of agar blocks is time consuming, it was found to be the best method for screening resistance to drop. Optimum Time to Evaluate Cultivars Since the degree of susceptibility among cultivars to lettuce drop varied with time, it was necessary to estab- lish the optimum time to evaluate cultivars. Resistance was measured by the cultivar's delay in succumbing to the disease. This is shown by number of days required for the cultivars to reach 50% mortality. When this criterion was applied to the 16 PI acces- sions and cultivars (Table 7), it was noted that the highest number of days for 50% mortality was 12.7. Therefore, dif- ferences in susceptibility among cultivars were compared using cumulative mortality data up to the 15th day following ino- culation. Statistical analysis showed that Grand Rapids, a cultivar grown in Michigan greenhouses, was one of the most susceptible. Cultivars found significantly different at the 34 5% level compared with Grand Rapids were PI 250427, Taiwan, PI 251790, PI 165063, PI 255568, Bibb and MSU 73-44. Greenhouse Ground Bed vs. Flats Culture A correlation coefficient of r = .77 was found be- tween the results of planting in the greenhouse bed and those planted in flats. This r value was significant at P = .001 suggesting that the results obtained by growing in the soil bed and flats were closely related. PART II. RESISTANCE TO LETTUCE DROP MATERIALS AND METHODS 1. Parental materials Since it is difficult to obtain 100 percent hybridi- zation in lettuce due to the nature of the reproductive mechanism, variable amounts of selfed progenies occur in the female parents when hybridized. Since the likelihood of self progenies occurring in the population is high, leaf color was used as a genetic marker. The green leaf trait, which is monogenic dominant to yellow-green leaf, was used as the male parents. No reciprocal crosses was made, since it would be difficult to distinguish the hybrids from the selfs if the green leaf color parent was used as the seed parent. The resistant parents selected for the genetic studies were derived from cultivars studied in the previous section (Figure 2),‘yig.: -PI 250427, and 4 selections from the cultivar Taiwan, designated as Taiwan-l, Taiwan-2, Taiwan-3, and Taiwan-4. All the parental material was allowed to self polli- nate. PI 250427 had a dense green foliage. The leaves were oval, with a length to wide ratio of 3:1. The leaf margins 35 36 were serrate, and the leaf blades were slightly crink- led. The 4 selections derived from the cultivar Taiwan were morphologically similar. The leaf form was oval, with a length to width ratio of 3:1 and leaf color was dark green. The susceptible parents for these studies were the cultivars Grand Rapids and PI 278110 (Figure 2). The cultivar Grand Rapids is a leaf lettuce grown commercially in greenhouses in Michigan. The length-to- width ratio of leaves is 2:1, shorter than the other culti- vars. The leaves are yellow-green with serrate margins. The cultivar Grand Rapids is late flowering and therefore bolting was induced by spraying 20 ppm gibberellic acid when the plants were at the 5 leaf stage. PI 278110 is a butterhead lettuce which has yellow- green leaves with a smooth leaf margin. 2. Pollination Pollination was done in the greenhouse, using a technique developed by Oliver (1910) and described by Newton and Sequeira (1972b). The petals of unopen flowers were cut with scissors approximately 2 cm from the tip. When the pollen laden stigma protruded a few hours later, it was washed by a jet stream of water. After the flower dried, they were pollinated. One male flower was used to pollinate 1-3 female flowers. Seeds matured approximately 12 days after pollina- tion. Hybridization was made in April, while backcrossing and selfing were in August and early September, 1979. 37 Figure 2: Parental materials: Top left: PI 278110 (susceptible); Top Right: PI 250427 (resistant): Bottom left: Grand Rapids (susceptible); Bottom Right: Taiwan (resistant) 39 One plant of PI 250427 and one plant from each of the 4 Taiwan cultivars were allowed to self to obtain par- ental seeds. F2 seeds were obtained by allowing the F1 plants to self. A random sample of alleged resistant plants showing Grand Rapids phenotype from the F2 population were selfed for the F3 generation (Table 9). The F2 population was obtained by selfing randomly selected alleged resistant plants from the F3 population. 3. Screening for Disease Resistance Seeds were sown in moist vermiculite in flats in the greenhouse. Seedlings were transplanted 7-10 days after sowing intoESx 5 cm peat pots containing artificial mixture medium. Seventy peat pots were placed per flat. Liquid fertilizer was applied 3 and 17 days following transplanting with 2 g of 20-20-20 (N-P-K) soluble fertilizer per liter of water. The greenhouse soil bed was sterilized prior to growing a standard lettuce crop. Following harvest the bed was used to grow the experimental materials. The soil was tilled 20 cm deep prior to planting. Tilling was repeated after the third planting. The soil was leveled using a hand rake before planting. S. sclerotiorum was grown at 20 C for 5 days on potatov dextrose agar (PDA) medium prior to inoculation. Plants were 40 TABLE 9: NUMBER OF THE F LINES SCREENED FOR RESISTANCE TO LETTUCE DROP 3 Parentage Number of Lines Grand Rapids x PI 250427 10 Grand Rapids x Taiwan-l 20 Grand Rapids x Taiwan-2 27 Grand Rapids x Taiwan-3 24 Grand Rapids x Taiwan-4 19 PI 278110 x PI 250427 8 PI 278110 x Taiwan-1 9 PI 278110 x Taiwan-2 11 PI 278110 x Taiwan-3 l4 inoculated by placing a 1/2 cm2 agar block culture 1 cm from the base of the 3-week-old seedlings one day prior to trans- planting into the greenhouse ground bed. The planting dis- stance in the bed was 15 x 15 cm. The experiments were in a completely randomized block design and replicated 3 times. The randomization was done within and between families for each of the plantings. Numbers of plants used in the experiments for each repli- cate were 10 plants for P1, P2 and F1, reciprocal backcrosses, and 50 plants for the F2 population. 30 plants for the 41 Total number of plants per family in each experiment were 30 plants for P1, P2 and F1, 90 plants for reciprocal back- crosses, and 150 plants for the F2 generation. The culti- vars Grand Rapids and PI 278110 were used as guard rows. Crosses involving Grand Rapids were tested in 5 different plantings during the fall of 1979 and winter 1980, while that of PI 278110 was done once in March 1980. The air temperature and relative humidity were recorded using a hygrothermograph placed 20 cm above soil level. Taylor Weather-Hawk recording thermometer number 2354 (Taylor In- strument Companies, Rochester, N.Y) was used to record the soil temperature (Table 10). The temperature sensing bulb was inserted 7 cm in the ground bed. The relative humidity of the greenhouse was maintained at 80% r 10%. The numbers of dead plants were recorded every 2 days starting from the 6th day and ending on the 18th day after transplanting. 4. Screening of the F3 Population Seedlings were grown in the greenhouse as described earlier for 19 days after transplanting, then transferred to the cold Chamber at the Horticultural Research Farm. The temperature in the cold chamber was 14 C and the rela- tive humidity was maintained at 80%i5%. Fluorescent lamps were used as the source of light. Plants were inoculated when the seedlings were 21 days old using the similar technique used for the F2 population. 42 TABLE 10: THE SOIL AND AIR TEMPERATURES IN THE GREENHOUSE ~ DURING EACH OF THE SIX PLANTINGS 1 2 3 4 5 6 Nov.l- Nov.24- Dec.22- Jan.l9- Febr. 3- March 1- Nov.20 Dec.17 Jan.ll Jan.29 Febr.26 March 21 Soil Temperature (°C): Mean 14 12 13 16 14 16 Range 10-17 9-16 11-16 13-19 11-19 11-24 Air Temperature (°C): Mean 15 16 14 14 12 13 Range 11-28 11-28 11-26 10-24 9-24 11-23 The plot consisted of 10 seedlings for P and P2,and l 20 seedlings for each line of F2 and F3, replicated 3 times in a completely randomized block design. A total of 3 plantings were made. Two plantings were materials hybrid- ized with Grand Rapids, while the third was those hybridized with PI 278110. Numbers of dead plants were recorded every 2 days beginning after the 6th day. Data were reported as percent survivors. 5. Screening of the F4 Population F4 seedlings were grown in the greenhouse similar to that described earlier. Twenty-one day old seeldings were inoculated using the method described for the F2 and'F3 popu- lations. The plot consisted of 7 seedlings for each P1 and 92. l4 seedlings for each F2 and F4, replicated 5 times in 43 a completely randomized design. Data were recorded every day following the 3rd day after inoculation; 6. Weighting of the Backcross Populations Difficulty in obtaining complete control in hybrid- ization of lettuce due to the nature of the reproductive mechanism made it necessary to formulate a weighting factor. Leaf color was used as a genetic market to identify the hybrids from the selfs. The magnitude of self pollination was found to be 30%. Prior to transplanting into the green- house bed, selfed seedlings were discarded from the‘Fl populations. Since selfed seedlings were not discernable from the BC to P (BCl) to BC to P2 (BCZ) seedlings, a l weighting factor was formulated to remove the selfs from the crossed seedlings. The presence of incomplete pene- trance in the resistance to lettuce drop was included in the weighting. The magnitude of this factor in the BCl is es- timated from that of the P1 population. The weighting (Grand Rapids or PI 278110 x F1) is as formula for the BCl follows: Rw = R0 - number of seedlings grown in BCl population x percentage of self-pollination x percentage of penetrance in P1 where Rw = weighted number of the resistant plants R0 = observed number of the resistant plants The weighting formula for BC2 (Fl X P2) was formulated as follows: 44 Since the F1 was used as the female parent, the penetration factor was based on the F2 segregating genera- tion. Rw= Ro - number of seedlings grown in BC2 population x percentage of self-pollination x percentage of penetrance in F2 7. Genetic Interpretation Resistance to lettuce drop was manifested by differ- ences in the length of time required for the plants to die and by differences in number of plants that survived on a given date for each cultivar. The resistant cultivars had a larger number of survivors at the greatest time to reach 50% mortality among cultivars. To establish the degree of resistance for the cultivars, the optimum time to evaluate the cultivars was based on the survivors from several ob- servations. The differences in the time to reach 50% mortality between the various generations were measured by using re- gression analysis. LSD test was applied to measure the significant differences in the number of survivors found between generations. The Chi-Square analysis for a 2 x 2 contingency table as described by Briggs and Knowles (1967) and Skory (1952) was applied to the parent materials in F2 tests. RESULTS AND DISCUSSION Parental Materials Grand Rapids seedlings were found to be the lowest in number of days following inoculation to reach 50% mor- tality among the parent lines tested (Table 11). The per- centage survivors of Grand Rapids and PI 278110 were signi-' ficantly lower (P a 0.05) than PI 250427 and the four Taiwan strains. This observation was similar to that reported in the previous section where 16 cultivars were tested. The results suggest that the four Taiwan strains possessed greater resistance to lettuce drop than Grand Rapids. In comparison to PI 250427 and the four Taiwan strains, PI 278110 showed a similarity in number when 50% of plants succumbed. Six days after inoculation, 10% of the PI 278110 seedlings survived as compared to more than 20% for PI 250427 and the four Taiwan strains. At the final observation, only 5% of PI 278110 seedlings survived, while those of PI 250427 and the four Taiwan strains showed more than 20% survivors (Appendix Table A-2). Pooling of the Data Bartlett's test for homogeneity of the five plantings of Grand Rapids x PI 250427 and Grand Rapids x four Taiwan 45 46 TABLE 11: NUMBER OF DAYS AFTER INOCULATION TO REACH 50% MORTALITY AND PERCENT SURVIVORS ON THE 6TH DAY FOLLOWING INOCULATION OF THE PARENT LINES Lines Number of days to Survivors 1) 50% mortality ‘ (%) Grand Rapids 4.6 Oa PI 278110 5.3 10a PI 250427 5.7 29b Taiwan-l 5.8 21b Taiwan-2 5.5 29b Taiwan-3 5.0 21b Taiwan-4 5.2 50b 1) Percentage within column followed by the same letter are not significantly different at the 5% level. 47 strains crosses did not show significant differences (P = 0.25 - 0.5), and therefore the data were pooled. The pooling of the data was based on the time of observation rather than the number of days after inoculation. The first observation was made, when approximately 10% of the seedlings of each planting died. Since the rate of mortality of each of the families was not similar, some of the families reached 50% mortality prior to the first observation. The number of days when 10% of the seedlings died varied from 5 to 8 days. In the first and second plantings it took 8 days, while in the fourth planting, 6 days, and in the third and fifth plantings, 5 days. Frequency distribution on the number of dead plants at each observation (Appendix Table A-3) showed that the four Taiwan strains in crosses with Grand Rapids and the three Taiwan strains in crosses with PI 278110 were not homo- geneous. Inheritance of Resistance Grand Rapids Crosses The time of obServation for 50% mortality for the Grand Rapids cultivar showed negative values in four out of five cases, while the other parental cultivars two of the parents showed negative values (Table 12); The data suggest that Grand Rapids succumbed earlier than the other cultivars. The negative values for the time of observation in- diated that more than 50% mortality occurred prior to the first observation. The occurrence of negative values was 48 due to the fact that the observation was made when 10% of the plants in the entire experimental plots succumbed, rather than on the mortality of the susceptible parent in each of the families. In the F1 generations of Grand Rapids crosses, the time of observation for 50% mortality was less than one, suggesting that the 50% mortality occurred prior to the second observation. In three of the crosses, the time of observation for 50% mortality for the F1 generation was greater than the parent mean. The time for 50% mortality in the F2 generations was foundtxnbe negative in four of the crosses, suggesting mortality in the F2 generation was observed to be sooner than in the F1 population. In four of the crosses, the backcrosses to the sup- posedly resistant parents resulted in larger values for time for 50% mortality than those values for backcrosses to the supposedly susceptible parent. The differences among the cultivars in survival per- centage were small (Table 13). Grand Rapids, the supposedly susceptible cultivar at the final observation showed total survival percentage of 48 to 62 percent. The supposedly resistant cultivars had survival percentages ranging from 57 to 63 percent. Survival percentages in the F1 populations were inter- mediate in three crosses, and lower than either parent in two of the crosses. The survival percentages in the F2 population 49 TABLE 12: TIME OF OBSERVATION TO REACH 50% MORTALITY FOR GRAND RAPIDS CROSSES Other Parents Generation PI 250427 Taiwan-l Taiwan-2 Taiwan-3 Taiwan-4 Grand Rapids -.2 Other Parents .8 Fl .2 F2 .6 BCl 1.6 BC2 .2 -.6 -1.7 .3 -l.8 .1 — .8 .3 —4.0*) .2 .5 .3 .5 -.7 -.7 -.7 .1.0 -.1 .0 .4 -1.5 -2.7*) -.1 -.7 .1.0 *) These figures did not measure the time of mortality for these generations, due perhaps to regression model used for the above data or random error. a muonoxumn some now own "mm you nonfiaommm omh unmouo sumo pom coaumnocom m can acmuma some now mmcwavmmm oma >Hmumefixoudam mums muons At 02 02 oz 02 02 02 mm? mm> 02 OZ Amoo " my oocmumwwaa ampcoumm 0 me mm em mm mm em em mm we Hm mom .5 mm em me mm we so he mm mm ee Hom em em mm mm mm me mm em mm me . Ne mm mm mm mm mm me em Ne em me He em as am me so me we be mm me hectare Lethe em as mm am «e we we em em em heedmm tempo Emma chm Emma ohm urea tum shad chm rhea chm elements muetzeha mnemzeme Huemzema emeomm He eoeumhmcdu mwcmumm umcuo Aswmmmomu mQHmmmmmO Bmcq Qz< QmHEB Eé m0<92m0mmm J<>H>mDm "ma mdmce 51 were also intermediate in three of the crosses, and lower than either parent in two of the crosses. In three of the crosses the percentage of the sur- vivors was higher in the backcross to the supposedly sus- ceptible Grand Rapids cultivar than in the backcrosses to the supposedly resistant other parents. PI 278110 crosses The time to reach 50% mortality in PI 278110 was less than that of the three Taiwan strains suggesting that PI 278110 was more susceptible than Taiwan strains (Table 14). In comparison with PI 250427, the time for 50% mortality was greater for PI 278110. The F1 generation (showed a higher value for the time to reach 50% mortality than both of the parents in two of the four crosses (PI 278110 x Taiwan 1 and PI 250427) and a lower value than either parents (PI 278110 and Taiwan-2) and a similar value in the cross PI 278110 x Taiwan-3. The F2 generation showed larger value in three of the crosses and lower than either parent in one of the crosses. The backcrosses to the supposedly resistent parent resulted in a lower value than that obtained from backcrosses to the supposedly susceptible parents. The survival percentage PI 278110 was significantly greater than that of supposedly resistant parents PI 250427 and Taiwan-l (Table 15). In the final observation PI 278110 showed higher survival percentage than Taiwan-2 by 13%, but 52 TABLE 14: TIME OF OBSERVATION FOR 50% MORTALITY FOR PI 278110 CROSSES Generation Other Parents PI 250427 Taiwan-l Taiwan-2 Taiwan-3 PI 278110 1.7 2.5 3.1 3.3 Other Parents 1.1 2.6 3.6 3.4 Fl 2.5 4.5 2.6 3.2 F2 4.0 3.1 3.3 3.7 BCl 3.5 1.0 2.0 3.7 BCZ 2.6 .0 1.9 3.1 was not significant (P a 0.05). When comparison was made between survival percentage of PI 278110 Taiwan-3, the latter cultivar showed 13% greater survival percentage, but not significant at the 5% level. F3 and F4 generations The observed mean survival percentages for the F3 and F4 generations in comparison to the parental means showed that a large percentage of survivors occurred in the F3 generation in all of Grand Rapids crosses observed in these two plantings (Table 16). These differences, how- ever, were not significantly different from the parents. In the F4 generation, two out of those 5 crosses mentioned in the F3 showed greater survival percentage over the parent means, but not significantly (P = 0.05). 53 TABLE 15: SURVIVAL PERCENTAGE AT THIRD AND LAST OBSERVATION FOR PI 278110 *) Other Parents PI 250427 Taiwan-l Taiwan-2 Taiwan-3 . Observations 3rd Last 3rd Last 3rd Last 3rd Last PI 278110 93 93 97 97 87 83 87 73 Other Parents 75 68 75 75 90 70 89 86 F1 93 190 97 87 83 80 93 87 F2 91 89 84 79 93 89 91 83 BC1 76 75 70 67 87 86 71 62 BC2 87 86 95 94 76 71 86 76 Parental Difference (P =.05) No Yes Yes Yes No No No No *) Seedlings raised for the test of each cross were ap- proximately 30 for each parent and the F1 generation, 150 for the F2 and 63 for each backcross. 54 V .mommnycouma CH czonm mum mcowamumcmm m can mm ca powwow modaa mo Hones: Hmuoar lmemas lmcmma lmemea -- -- me mad mNH vma 111 111 m ooa cod ocH 111 111 names Hmucoumm "mummy cowumuocomvm laced leaceea Asa.-ea legend -- we voa Ha mma add 111 m ooa ooa ooa coa 111 memos Hmpcoumm "Ocaucmam Ccoomm m Aoavhma AOHVHNH AoHVNm .oavmma «Acavmma mm mma vHH em Hma flea m coa ooa OOH cod cod mcmme Hmuconmm "mafiucmaa Human "mummy cofipmumcmo mm vncmzflme Mlcmzfime mncm3fim9 alcmzflme hmqomm Hm codpmuocoo mucoumm Honuo mood I mzamz adezmmad .mmmmomom maHdcm ozamo mom .NE oze mzH>m=m mzam: "SH mamas 55 The F3 generations of PI 278110 crosses showed a larger mean for survival percentage than the parental means in 2 of the 4 crosses, but the differences was not signifi- cant (Table 17). No change in survival percentage was ob- served in the F4 generation of these crosses. When survival percentage for individual lines was considered, several F3 lines were significantly greater (P . .05) than parental means (Table 18). In crosses in- volving Grand Rapids, nine out of 100 F3 lines tested showed significantly greater survival percentage, while crosses involving PI 278110 only one out of 42 lines tested was significant. In the F4 generations, none of the eight lines of the Grand Rapids crosses and 11 lines of PI 278110 crosses showed significantly greater in survival percentage than that of the parents. Interpretation of the Data It is difficult to explain the results genetically. The most likely explanation is that the time for 50% mortal- ity and survival percentage in all populations were primarily under the control of environment, while the genetic contri- bution to the variation for resistance was low. The varia- tion is discernable especially between the parents, since they were included in each planting. Low genetic variation for resistance was observed based on the following: 56 AND F LINES COM- TABLE 17: MEANS SURVIVAL PERCENTAGES or F3 2, FOR PI 278110 PARED TO THE PARENTAL MEANS AND F CROSSES. PARENTAL MEANS a 100% Other parents Generation PI 250427 Taiwan-1 Taiwan-2 Taiwan-3 F3 Generation Test: Parental MMeans 100 100 100 100 F2 91 89 102 103 F3 89(8)* 104(9) 113(11) 95(14) F4 Generation Test: Parental Means --- 100 100 --- F2 --- 98 76 --- F4 --- 95(5) 80(6) --— * Total number of lines tested in F3 and F4 generations are shown in parentheses. TABLE 18: NUMBER OF LINES THE F3 AND F GENERATIONS HAVING SIGNIFICANTLY GREATER SURVIVAL PERCENTAGE THAN THAT OF THE PARENT MEANS. (TOTAL NUMBER OF LINES TESTED IN EACH GENERATION SHOWN IN PARENTHESES). Other Parents Genera- PI Taiwan Taiwan Taiwan Taiwan tion 250427 -1 -2 -3 -4 Grand Rapids Crosses F3 2 (10) 2 (20) 0(27) 1(24) 3 (19) F4 -- -- 0(2) 0(3) 0 (3) PI 278110 Crosses F3 0 (a) 1 (9) 0(11) 0(14) -- r4 -- o (5) 0(5) -- —— 57 (1) Variation in survival percentage was observed for the parents. (2) Genetic gains were evident in the F3 generation when a large number of lines were sampled, while only an insignificant gain was observed when fewer of lines were sampled in the F4 generation. This suggested that the resistance to lettuce drop in the materials under study was polygenically controlled with low level of penetrance. CONCLUSIONS Data on inheritance of resistance to lettuce drop reported here were based on 14352 seedlings which included parents, F1, F2 and backcross generations grown in the greenhouse ground bed. In addition, results obtained were supported by data from performance of 10,140 F3 plants and 2030 F4 plants grown in flats. The level of penetrance for resistance to lettuce drop varied with time of observation.- Resistance was manifested by the delay in time of death from the pathogen and by the greater number of survivors. The level of penetrance was modified by environmental conditions, such as fluctuation of the soil and air temperatures ob- served. Penetrance level was also modified by the inoculum load since the soil was not sterilized between planting the first and the third planting, and the fourth and the sixth planting. The variation in penetrance level was discernable especially with the parents since they were included in each of the plantings. Genetic gain was observed when a large number of F3 lines were tested. No genetic gain was observed in the F4 since fewer number of lines were tested. These suggested that the resistance to lettuce drop in the materials under study was polygenically controlled with low level of penetrance. 58 59 It was observed that in some of the plantings the difference in the level of penetrance between two parents was not significant. This suggests that there is a possi- bility of selecting for resistance strains from the Grand Rapids cultivar. Robinson (1980) suggested since trans- ferring polygenically inherited Characters is difficult, selection can start from susceptible parents simultaneously with other desirable characters. A selection that began from low level of resistance has been demonstrated success- ful in breeding red clover resistant to Sclerotinia trifoliorum by Frandsen (1946). An increase from 4.6% to 67.3% of level of resistance was achieved in 6 years of selection. In the Grand Rapids cultivar the lowest level of penetrance for resistance at the final observation was 3%. APPENDIX 60 1amwcofim no: mum kuuoa wean .lummu m.>mx:ev edema am an» um succeeded Sancho gnu >n cmonHow CEEHOO m Canvas mommucmouoa Hm>fl>usm Ar nee ammm nm- mmm obese mme the: enema onmmm muons- onmee unwed tcmHmH meshed onem mem mttmm teem onem Shem enema Baum unmem tmm obese moo deflatedms onmsw tonam mood- onmee oped momEEH Emma Emma nemN enema use mcedmm hence umHm>HHH30 onmmm oee mmemm cove unwed ee-me am: ohm mam ems pee does creams "tweed ocecmmpm am: onmmm mee- mmooem code obese memmmm He ohm mom mam dose obese omsemm He Emma nemm tandem seem obese mmeemm He onmmm hem deem poem coed emeemm He and nemm moon-m mom has memeow He Emma Emma nemm mom was amused He to tea mes the obese Ame-ma He unmmN o-e mtos- obese unwed meomee He “OCOmeOUOm Hm Hm>fl>usm Mo R me me we a e mhm>euaso cowumasoocH acaonaom.m>ma .4 onaaqaoozH oszossoR msca Lo mmmzaz sexmsmm ac nmsmmmmo mm<>Heqso we «on mueezmommd q¢>H>mam .H-c mamas 61 TABLE A-2 SURVIVAL PERCENTAGES OF THE PARENT MATERIALS OBSERVED AT DIFFERENT NUMBER OF DAYS FOLLOWING INOCULATION Number of days following inoculation Cultivars 3 4 5 6 7 8 10 % of Survival Grand Rapids 86 57 '32 25 18 0 0 PI 278110 95 76 43 29 29 10 5 PI 250427 96 82 71 61 54 29 29 Taiwan-1 96 86 82 54 39 21 21 Taiwan-2 ' 100 79 71 so 29 ' 29 29 Taiwan-3 93 71 57 50 21 21 21 Taiwan-4 100 86 71 57 57 so so. 62 N ne.~ ee.~ e e n e e ee ee Rea sen ad oh oe -.~ An.a e e a e ea en es «me man e as we ~n.~ ea." e e e~ es «M ed. een an” amp at -.~ -.~ e a A a - ee ee ee nee e e e~.~ en.a A a A A a ea a. -e ems A . e. e-eezeea ~e.~ em.~ e e e - A be A. ee emu . e. avenue peace v-cmzues x evade: cameo -.~ me.~ e e a ea ea n- he see man we or oe e~.~ ~e.~ e e m A ee he ee -e~ can e cu we -.a ee.~ a e e ma em eea emu «en ems Re mm.~ e~.~ a e H - as me .e we deg e e -.~ ee.~ e a e m e ee «A «e eve . . e. m-eezeee -.~ -.~ a m A m ea ea ae as eve . e. negate tempo m-cmzuab x acumen pcuuo ep.e em.~ e e e e A e- ee eve ean we or oe -.~ -.~ A e e m «a e- e- sea ean e as we -.~ me. e n nu ma en ea one wen men at ~e.~ he.~ e e a m A AA ee ee sea A e ee.e De.~ e e e v A ee an em e-a a . e. e-eezees e~.~ ee.~ e e e e m e an em emu e. oedema deuce «nausea? x acume¢.pcaco m~.~ me." e n n A -~ Re eea ems dam we or oe an.~ ee.~ e A e e he an AA med nan e o» e ~e.~ e~.~ e A AA me do see ope men one Le -.~ ee.~ e e v A m as an ee meg A e e~.~ ee.~ e e e n e - em he «he a . e. e-etSRea e~.~ ee.~ e a A AA n as m. as awe . e. heedee canto Anccsqna x enemas Ucauo a~.a ee.~ e e es e As me as use pen me oh oe e~.~ e-.~ e e A e on em he one e.m e as we en.~ m~.~ ~ e as e~ ee as see eon ems Re em.~ e~.~ a a e e m we ea -e med e e -.~ ne.~ e e e e - ve me «e and a . e. seveee He ~e.~ me.a e e N m e ea em ee eve . e. negate cameo reamed name no Leena: seeeme He.x deedee etude em coo: e e m c m e z necewe than no «Lease do coeeecteoo cadua>uomno mo clam Ron‘s: «mace Donia: quack .mOHm<¢ QZ<¢O OZH>JO>2H mmmmOzU m toga ZOHB¢>¢mmmO m0 mmtnh Ezmzflmmmn kc m92uouno mo Olah no basis: deuce Aunts: Aqua? 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